Avian Reticuloendotheliosis Viral Oncogene Related B Regulates Lymphatic Endothelial Cells during Vessel Maturation and Is Required for Lymphatic Vessel Function in Adult Mice.

NF-κB signals through canonical transcription factor p65 (RelA)/p50 and noncanonical avian reticuloendotheliosis viral oncogene related B (RelB)/p52 pathways. The RelA/p50 is involved in basal and inflammatory lymphangiogenesis. However, the role of RelB/p52 in lymphatic vessel biology is unknown. Herein, we investigated changes in lymphatic vessels (LVs) in mice deficient in noncanonical NF-κB signaling and the function of RelB in lymphatic endothelial cells (LECs). LVs were examined in Relb-/-, p52-/-, or control mice, and the gene expression profiles in LECs with RelB knockdown. Relb-/-, but not p52-/-, mice exhibited multiple LV abnormalities. They include the following: i) increased capillary vessel diameter, ii) reduced smooth muscle cell (SMC) coverage of mature vessels, iii) leakage, and iv) loss of active and passive lymphatic flow. Relb-/- mature LVs had thinner vessel walls, more apoptotic LECs and SMCs, and fewer LEC junctions. RelB knockdown LECs had decreased growth, survival, and adhesion, and dysregulated signaling pathways involving these cellular events. These results suggest that Relb-/- mice have abnormal LVs, mainly in mature vessels with reduced SMC coverage, leakage, and loss of contractions. RelB knockdown in LECs leads to reduced growth, survival, and adhesion. RelB plays a vital role in LEC-mediated LV maturation and function.

Innate Immune Cells Are Regulated by Axl in Hypertensive Kidney.

The balance between adaptive and innate immunity in kidney damage in salt-dependent hypertension is unclear. We investigated early renal dysfunction and the influence of Axl, a receptor tyrosine kinase, on innate immune response in hypertensive kidney in mice with lymphocyte deficiency (Rag1-/-). The data suggest that increased presence of CD11b+ myeloid cells in the medulla might explain intensified salt and water retention as well as initial hypertensive response in Rag1-/- mice. Global deletion of Axl on Rag1-/- background reversed kidney dysfunction and accumulation of myeloid cells in the kidney medulla. Chimeric mice that lack Axl in innate immune cells (in the absence of lymphocytes) significantly improved kidney function and abolished early hypertensive response. The bioinformatics analyses of Axl-related gene-gene interaction networks established tissue-specific variation in regulatory pathways. It was confirmed that complement C3 is important for Axl-mediated interactions between myeloid and vascular cells in hypertensive kidney. In summary, innate immunity is crucial for renal dysfunction in early hypertension, and is highly influenced by the presence of Axl.

Effect of gabapentin on sexual function in vulvodynia: a randomized, placebo-controlled trial.

BACKGROUND: Sexual dysfunction is common in women with vulvodynia. OBJECTIVE: The purpose of this study was (1) to evaluate whether extended-release gabapentin is more effective than placebo in improving sexual function in women with provoked vulvodynia and whether there is a relationship between treatment outcome and pelvic pain muscle severity that is evaluated by palpation with standardized applied pressure and (2) to evaluate whether sexual function in women with provoked vulvodynia would approach that of control subjects who report no vulvar pain either before or after treatment. STUDY DESIGN: As a secondary outcome in a multicenter double-blind, randomized crossover trial, sexual function that was measured by the Female Sexual Function Index was evaluated with gabapentin (1200-3000 mg/d) compared with placebo. Pain-free control subjects, matched by age and race, also completed Female Sexual Function Index for comparison. RESULTS: From August 2012 to January 2016, 230 women were screened at 3 academic institutions, and 89 women were assigned randomly to treatment. Gabapentin was more effective than placebo in improving overall sexual function (adjusted mean difference, 1.3; 95% confidence interval, 0.4-2.2; P=.008), which included desire (mean difference, 0.2; 95% confidence interval, 0.0-3.3; P=.04), arousal (mean difference, 0.3; 95% confidence interval, 0.1-0.5; P=.004), and satisfaction (mean difference, 0.3; 95% confidence interval, 0.04-0.5; P=.02); however, sexual function remained significantly lower than in 56 matched vulvodynia pain-free control subjects. There was a moderate treatment effect among participants with baseline pelvic muscle pain severity scores above the median on the full Female Sexual Function Index scale (mean difference, 1.6; 95% confidence interval, 0.3-2.8; P=.02) and arousal (mean difference, 0.3; 95% confidence interval, 0.1-0.6; P=.01) and pain domains (mean difference, 0.4; 95% confidence interval, 0.02-0.9; P=.04). CONCLUSION: Gabapentin improved sexual function in this group of women with provoked vulvodynia, although overall sexual function remained lower than women without the disorder. The most statistically significant increase was in the arousal domain of the Female Sexual Function Index that suggested a central mechanism of response. Women with median algometer pain scores >5 improved sexual function overall, but the improvement was more frequent than the pain domain. We hypothesize that gabapentin may be effective as a pharmacologic treatment for those women with provoked vulvodynia and increased pelvic muscle pain on examination.

Endocrine disruptor bisphenol A is implicated in urinary voiding dysfunction in male mice.

Estrogens, acting synergistically with androgens, are known from animal experiments to be important in lower urinary tract symptoms (LUTS) and benign prostate enlargement. Human exposure to environmental estrogens occurs throughout the life span, but the urologic health risks in men are largely unknown. Bisphenol A (BPA) is an endocrine disruptor implicated in male urogenital malformations. Given the role of estrogens in male LUTS, we studied the effects of BPA administered in combination with testosterone (T) on the urinary voiding behavior of adult male mice. Adult male mice underwent subcutaneous implantation with slow-release pellets of 25 mg BPA or 2.5 mg estradiol-17ß (E2), plus 25 mg T, and were compared with untreated (UNT) mice that underwent sham surgery. We studied urinary voiding behavior noninvasively for 1 mo before treatment and for 4 mo after treatment. After euthanasia, we evaluated bladder volume and mass. Mice treated with T+BPA had increased bladder volume ( P < 0.05) and mass ( P < 0.01) compared with UNT mice. After 4 mo of treatment with T+BPA, three of five mice developed voiding dysfunction in the form of droplet voiding or an intermediate pattern of voiding different from both UNT and T+E2-treated mice. Treatment of male mice with BPA or estradiol induces voiding dysfunction that manifests at later time points, implicating the endocrine disruptor, BPA, as a contributor to male LUTS.

Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates Osteoclast Differentiation via the Ca2+ /NFATc1 Axis.

DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion between osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function. The absence of a known DC-STAMP ligand, however, has hindered the elucidation of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. Decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of the tyrosine residues surrounding the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that the ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1/Ca2+ axis. J. Cell. Physiol. 232: 2538-2549, 2017. © 2016 Wiley Periodicals, Inc.

Relationship between nongenital tender point tenderness and intravaginal muscle pain intensity: ratings in women with provoked vestibulodynia and implications for treatment.

BACKGROUND: Vulvodynia is a chronic vulvar pain disorder and fibromyalgia is a chronic widespread musculoskeletal pain disorder, both of unknown etiology. Association of these conditions is well documented. Intravaginal algometer measurement of tenderness to pressure applied to the pelvic floor muscles helps define vulvodynia associated with musculoskeletal factors. Women with both vulvodynia and fibromyalgia might have increased pelvic muscle pain compared to women with vulvodynia alone, defining the possible link of these 2 conditions. OBJECTIVE: We sought to: (1) correlate pain intensity during the nongenital tender point tenderness examination to pain intensity with the vaginal algometer in women with provoked vestibulodynia, and (2) determine whether subjects with provoked vestibulodynia and fibromyalgia had higher pain intensity scores with the vaginal algometer than those without fibromyalgia. STUDY DESIGN: In all, 92 subjects referred for vulvar pain were confirmed to have provoked vestibulodynia using the cotton swab test. A diagnosis of fibromyalgia was made if pain was present (numeric rating scale >1) in at least 11 sites of the 18-point nongenital tender point tenderness exam. Vaginal pain sensitivity was measured using an intravaginal pressure algometer, where 0.1, 0.3, and 0.5 kg/cm2 forces were applied digitally in random assignment by force and location to the right and left iliococcygeus muscle regions and the posterior vaginal wall. Both tender point tenderness and algometer pain intensity were reported on a 0 (no pain) to 10 (worse pain) numeric rating scale. Correlations were computed between the composite pain intensity (total of rating scale from each pressure threshold at specified site) of nongenital and those of iliococcygeus regions and the posterior vaginal wall. Independent t tests were used to determine differences in iliococcygeus regions and the posterior vaginal algometer pain ratings and presence or absence of fibromyalgia. The significance level was at P < .05. The data were expressed as mean ± SD. RESULTS: A significant correlation was found between numeric rating scale pain scores on the nongenital tender point tenderness exam and algometer testing on the iliococcygeus region (r = 0.44, P < .0001) and the posterior vaginal wall (r = 0.45, P < .0001). Subjects with fibromyalgia by tender point tenderness had significantly higher iliococcygeal pain (6.14 ± 2.07 vs 3.74 ± 2.22, P = .0001) and posterior vaginal wall pain (5.67 ± 2.10 vs 3.07 ± 2.16, P < .0001) than women without fibromyalgia by tender point tenderness. CONCLUSION: Women with provoked vestibulodynia who experience more severe pain with nongenital tender point palpation also experience more deep vaginal pain on pelvic exam. Those who fulfill the diagnosis of fibromyalgia show significantly more intense deep vaginal pain to palpation of iliococcygeus muscles and posterior vaginal wall. Further research using a more precise definition of fibromyalgia is necessary to confirm this relationship, but findings suggest that women with provoked vestibulodynia coexisting with fibromyalgia have greater risk of superimposed vaginal muscle pain and may be candidates for early adjunctive pelvic floor physical therapy. These findings need to be explored in women with generalized, nonprovoked vulvodynia.

Estrogen receptor-α is a key mediator and therapeutic target for bladder complications of benign prostatic hyperplasia.

PURPOSE: Estrogens are important in prostate growth and have a role in benign prostatic hyperplasia. However, to our knowledge no current therapy directly targets estrogen action. Estrogens act primarily via estrogen receptors α and ß. In a mouse model we evaluated the relative contribution of these receptors to bladder complications of benign prostatic hyperplasia. We also evaluated the prevention of these bladder complications using the selective estrogen receptor modulators raloxifene and tamoxifen (estrogen receptor-α selective antagonists), and R,R-THC (estrogen receptor-ß selective antagonist). MATERIALS AND METHODS: Adult male C57bl/6 mice received implants of 25 mg testosterone and 2.5 mg 17ß-estradiol slow release pellets. Untreated controls underwent sham surgery. We evaluated the contributions of the estrogen receptor subtypes in ERαKO and ERßKO mice compared to their respective wild-type litter mates. Wild-type mice treated with testosterone plus 17ß-estradiol were compared to mice treated with testosterone plus 17ß-estradiol and 25 mg selective estrogen receptor modulators to evaluate the prevention of benign prostatic hyperplasia complications by selective estrogen receptor modulators. RESULTS: Large bladders with urinary retention developed in ERαWT and ERßWT litter mates treated with testosterone plus 17ß-estradiol but such bladders did not develop in ERαKO mice treated with testosterone plus 17ß-estradiol. ERßKO mice treated with testosterone plus 17ß-estradiol had large bladders with urinary retention and increased bladder mass. Cotreatment with the estrogen receptor-α antagonist raloxifene resulted in decreased bladder mass compared to that in wild-type mice treated with testosterone plus 17ß-estradiol. Bladders in mice treated with the estrogen receptor-ß antagonist R,R-THC were similar to those in testosterone plus 17ß-estradiol treated mice. CONCLUSIONS: Estrogen receptor-α but not ß is a key mediator of bladder complications of benign prostatic hyperplasia and a potential target for future therapies.

Quantitative volumetric imaging of normal, neoplastic and hyperplastic mouse prostate using ultrasound.

BACKGROUND: Genetically engineered mouse models are essential to the investigation of the molecular mechanisms underlying human prostate pathology and the effects of therapy on the diseased prostate. Serial in vivo volumetric imaging expands the scope and accuracy of experimental investigations of models of normal prostate physiology, benign prostatic hyperplasia and prostate cancer, which are otherwise limited by the anatomy of the mouse prostate. Moreover, accurate imaging of hyperplastic and tumorigenic prostates is now recognized as essential to rigorous pre-clinical trials of new therapies. Bioluminescent imaging has been widely used to determine prostate tumor size, but is semi-quantitative at best. Magnetic resonance imaging can determine prostate volume very accurately, but is expensive and has low throughput. We therefore sought to develop and implement a high throughput, low cost, and accurate serial imaging protocol for the mouse prostate. METHODS: We developed a high frequency ultrasound imaging technique employing 3D reconstruction that allows rapid and precise assessment of mouse prostate volume. Wild-type mouse prostates were examined (n = 4) for reproducible baseline imaging, and treatment effects on volume were compared, and blinded data analyzed for intra- and inter-operator assessments of reproducibility by correlation and for Bland-Altman analysis. Examples of benign prostatic hyperplasia mouse model prostate (n = 2) and mouse prostate implantation of orthotopic human prostate cancer tumor and its growth (n = ) are also demonstrated. RESULTS: Serial measurement volume of the mouse prostate revealed that high frequency ultrasound was very precise. Following endocrine manipulation, regression and regrowth of the prostate could be monitored with very low intra- and interobserver variability. This technique was also valuable to monitor the development of prostate growth in a model of benign prostatic hyperplasia. Additionally, we demonstrate accurate ultrasound image-guided implantation of orthotopic tumor xenografts and monitoring of subsequent tumor growth from ~10 to ~750 mm(3) volume. DISCUSSION: High frequency ultrasound imaging allows precise determination of normal, neoplastic and hyperplastic mouse prostate. Low cost and small image size allows incorporation of this imaging modality inside clean animal facilities, and thereby imaging of immunocompromised models. 3D reconstruction for volume determination is easily mastered, and both small and large relative changes in volume are accurately visualized. Ultrasound imaging does not rely on penetration of exogenous imaging agents, and so may therefore better measure poorly vascularized or necrotic diseased tissue, relative to bioluminescent imaging (IVIS). CONCLUSIONS: Our method is precise and reproducible with very low inter- and intra-observer variability. Because it is non-invasive, mouse models of prostatic disease states can be imaged serially, reducing inter-animal variability, and enhancing the power to detect small volume changes following therapeutic intervention.

In vivo quantification of lymph viscosity and pressure in lymphatic vessels and draining lymph nodes of arthritic joints in mice.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF-Tg mice, a model of inflammatory-erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre-arthritic 'expanding' phase, and then 'collapses' with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild-type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP-FRAP) of injected FITC-BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P < 0.05). Direct measurement of intra-lymph node pressure (LNP) demonstrated a decrease in expanding PLN versus WT pressure (3.41 ± 0.43 vs. 6.86 ± 0.56 cmH2O; P < 0.01), which dramatically increased to 9.92 ± 1.79 cmH2O in collapsed PLN. Lymphatic pumping pressure (LPP), measured indirectly by slowly releasing a pressurized cuff occluding indocyanine green (ICG), demonstrated an increase in vessels afferent to expanding PLN versus WT (18.76 ± 2.34 vs. 11.04 ± 1.47 cmH2O; P < 0.01), which dropped to 2.61 ± 0.72 cmH2O (P < 0.001) after PLN collapse. Herein, we document the first in vivo measurements of murine lymph viscosity and lymphatic pressure, and provide evidence to support the hypothesis that lymphangiogenesis and lymphatic transport are compensatory mechanisms to prevent synovitis via increased drainage of inflamed joints. Furthermore, the decrease in lymphatic flow and loss of LPP during PLN collapse are consistent with decreased drainage from the joint during arthritic flare, and validate these biomarkers of RA progression and possibly other chronic inflammatory conditions.

Efficacy of B cell depletion therapy for murine joint arthritis flare is associated with increased lymphatic flow.

OBJECTIVE: B cell depletion therapy ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritis flare in tumor necrosis factor (TNF)-transgenic mice is associated with efferent lymph node (LN) "collapse," triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. The aim of this study was to examine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells. METHODS: We used contrast-enhanced magnetic resonance imaging, indocyanine green near-infrared imaging, and intravital immunofluorescence imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-transgenic mice. We conducted tests to determine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic draining and cell egress from arthritic joints. RESULTS: Unlike active lymphatics to normal and prearthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescence imaging demonstrated that CD11b+ monocyte/macrophages in lymphatic vessels afferent to expanding LNs travel at high velocity (mean±SD 186±37 µm/second), while these cells are stationary in lymphatic vessels afferent to collapsed popliteal LNs. B cell depletion therapy for arthritis flares in TNF-transgenic mice significantly decreased knee synovium volume (by 50% from the baseline level) and significantly increased lymphatic clearance compared with placebo (P<0.05). This increased lymphatic drainage restored macrophage egress from inflamed joints without recovery of the lymphatic pulse. CONCLUSION: These results support a novel mechanism in which B cell depletion therapy for joint arthritis flares lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.

KDM3B Single-Nucleotide Polymorphisms Impact Radiation Therapy Toxicity Through Circular RNA-Mediated KDM3B Expression and Inflammatory Responses.

PURPOSE: Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) associated with radiation therapy (RT) toxicities in patients with prostate cancer. SNP rs17599026 in intron 21 of KDM3B is significantly associated with the development of late urinary toxicity, specifically in the increase in urinary frequency 2 years after RT compared with pretreatment conditions. The present study aimed to provide mechanistic insights for this association. METHODS AND MATERIALS: Using human tissues and cell lines, we examined the protein expression of KDM3B and molecular mechanisms underlying the SNP modulation by variants of KDM3B SNP alleles. In animals with normal and heterozygous expressions of Kdm3b, we examined the relationship between Kdm3b expression and radiation toxicity. RESULTS: KDM3B rs17599026 lies in a motif important for circular RNA expression that is responsible for sponging miRNAs to regulate KDM3B expression. Using a murine model with heterozygous deletion of the Kdm3b gene, we found that lower Kdm3b expression is associated with altered pattern of urination after bladder irradiation, which is related to differential degrees of tissue inflammation as measured by analyses of gene expression, lymphocyte infiltration, and noninvasive ultrasound imaging. CONCLUSIONS: KDM3B SNPs can impact its expression through regulating noncoding RNA expression. Differential KDM3B expression underlies radiation toxicity through tissue inflammation at the molecular and physiological level. Our study outcome offers a foundation for mechanism-based mitigation for radiation toxicity for prostate cancer survivors.

Inflammation, lipid dysregulation, and transient receptor potential cation channel subfamily V member 4 signaling perpetuate chronic vulvar pain.

ABSTRACT: Localized provoked vulvodynia is characterized by chronic vulvar pain that disrupts every aspect of the patient's life. Pain is localized to the vulvar vestibule, a specialized ring of tissue immediately surrounding the vaginal opening involved in immune defense. In this article, we show inflammation is the critical first step necessary for the generation of pain signals in the vulva. Inflammatory stimuli alone or combined with the transient receptor potential cation channel subfamily V member 4 (TRPV4) agonist 4α-phorbol 12,13-didecanoate stimulate calcium flux into vulvar fibroblast cells. Activity is blocked by the TRPV4 antagonist HC067047, denoting specificity to TRPV4. Using lipidomics, we found pro-resolving lipids in the vulvar vestibule were dysregulated, characterized by a reduction in pro-resolving mediators and heightened production of inflammatory mediators. We demonstrate specialized pro-resolving mediators represent a potential new therapy for vulvar pain, acting on 2 key parts of the disease mechanism by limiting inflammation and acutely inhibiting TRPV4 signaling.

Near-Infrared Imaging of Indocyanine Green Identifies Novel Routes of Lymphatic Drainage from Metacarpophalangeal Joints in Healthy Human Hands.

Background: Collecting lymphatic vessel (CLV) dysfunction has been implicated in various diseases, including rheumatoid arthritis (RA). RA patients with active hand arthritis exhibit significantly reduced lymphatic clearance of the web spaces adjacent to the metacarpophalangeal (MCP) joints and a reduction in total and basilic-associated CLVs on the dorsal surface of the hand by near-infrared (NIR) imaging of indocyanine green (ICG). In this pilot study, we assessed direct lymphatic drainage from MCP joints and aimed to visualize the total lymphatic anatomy using novel dual-agent relaxation contrast magnetic resonance lymphography (DARC-MRL) in the upper extremity of healthy human subjects. Methods and Results: Two healthy male subjects >18 years old participated in the study. We performed NIR imaging along with conventional- or DARC-MRL following intradermal web space and intra-articular MCP joint injections. ICG (NIR) or gadolinium (Gd) (MRL) was administered to visualize the CLV anatomy of the upper extremity. Web space draining CLVs were associated with the cephalic side of the antecubital fossa, while MCP draining CLVs were localized to the basilic side of the forearm by near-infrared indocyanine green imaging. The DARC-MRL methods used in this study did not adequately nullify the contrast in the blood vessels, and limited Gd-filled CLVs were identified. Conclusion: MCP joints predominantly drain into basilic CLVs in the forearm, which may explain the reduction in basilic-associated CLVs in the hands of RA patients. Current DARC-MRL techniques show limited identification of healthy lymphatic structures, and further refinement in this technique is necessary. Clinical trial registration number: NCT04046146.

Central and peripheral tau retention modulated by an anti-tau antibody.

Tau protein blood levels dependent on its distribution to peripheral organs and possible elimination from the body. Thus, the peripheral distribution of CSF-derived tau protein was explored, especially since there is a transition to blood-based biomarkers and the emerging idea that tau pathology may spread beyond brain. Near infrared fluorescence (NIRF) was mainly used to analyze tau (tau-NIRF) distribution after its intracisternal or intravenous injection. There was a striking uptake of blood- or CSF-derived tau-NIRF protein by the skeletal structures, liver, small intestine (duodenum), gall bladder, kidneys, urinary bladder, lymph nodes, heart, and spleen. In aging and in older APP/PS1 mice, tau uptake in regions, such as the brain, liver, and skeleton, was increased. In bone (femur) injected tau protein was associated with integrin-binding sialoprotein (IBSP), a major non-collagenous glycoprotein that is associated with mineralization. Tau-NIRF was cleared slowly from CSF via mainly across the cribriform plate, and cervical lymph nodes. In brain, some of the CSF injected tau protein was associated with NeuN-positive and PDGFRý-positive cells, which may explain its retention. The presence of tau in the bladders suggested excretion routes of tau. CSF anti-tau antibody increased CSF tau clearance, while blood anti-tau antibody decreased tau accumulation in the femur but not in liver, kidney, and spleen. Thus, the data show a body-wide distribution and retention of CSF-derived tau protein, which increased with aging and in older APP/PS1 mice. Further work is needed to elucidate the relevance of tau accumulation in each organ to tauopathy.

Implementation of automated behavior metrics to evaluate voluntary wheel running effects on inflammatory-erosive arthritis and interstitial lung disease in TNF-Tg mice.

BACKGROUND: Although treatment options and algorithms for rheumatoid arthritis (RA) have improved remarkably in recent decades, there continues to be no definitive cure or pharmacologic intervention with reliable long-term efficacy. For this reason, the combination of medications and healthy lifestyle modifications are essential for controlling joint disease, and extra-articular manifestations of RA, such as interstitial lung disease (ILD) and other lung pathologies, which greatly impact morbidity and mortality. Generally, exercise has been deemed beneficial in RA patients, and both patients and clinicians are motivated to incorporate effective non-pharmacologic interventions. However, there are limited evidence-based and specific exercise regimens available to support engagement in such activities for RA patients. Here, we provided the continuous opportunity for exercise to mice and implemented automated recording and quantification of wheel running behavior. This allowed us to describe the associated effects on the progression of inflammatory-erosive arthritis and ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA. METHODS: Wild-type (WT; males, n=9; females, n=9) and TNF-Tg (males, n=12; females, n=14) mice were singly housed with free access to a running wheel starting at 2 months until 5 to 5.5 months of age. Measures of running included distance, rate, length, and number of run bouts, which were derived from continuously recorded data streams collected automatically and in real-time. In vivo lung, ankle, and knee micro-computed tomography (micro-CT), along with terminal micro-CT and histology were performed to examine the association of running behaviors and disease progression relative to sedentary controls. RESULTS: TNF-Tg males and females exhibited significantly reduced running distance, rate, length, and number of run bouts compared to WT counterparts by 5 months of age (p<0.0001). Compared to sedentary controls, running males and females showed increased aerated lung volumes (p<0.05) that were positively correlated with running distance and rate in female mice (WT: Distance, ρ=0.705/rate, ρ=0.693 (p<0.01); TNF-Tg: ρ=0.380 (p=0.06)/ρ=0.403 (p<0.05)). Talus bone volumes were significantly reduced in running versus sedentary males and negatively correlated with running distance and rate in TNF-Tg mice (male: ρ=-903/ρ=-0.865; female: ρ=-0.614/ρ=-0.594 (p<0.001)). Histopathology validated the lung and ankle micro-CT findings. CONCLUSIONS: Implementation of automated wheel running behavior metrics allows for evaluation of longitudinal activity modifications hands-off and in real-time to relate with biomarkers of disease severity. Through such analysis, we determined that wheel running activity increases aerated lung volumes, but exacerbates inflammatory-erosive arthritis in TNF-Tg mice. To the end of a clinically relevant model, additional functional assessment of these outcomes and studies of pain behavior are warranted.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Vascular endothelial growth factor C attenuates joint damage in chronic inflammatory arthritis by accelerating local lymphatic drainage in mice.

OBJECTIVE: To investigate whether the enhancement of joint lymphangiogenesis by injection of vascular endothelial growth factor C (VEGF-C) adeno-associated virus (AAV) into the affected joints has therapeutic efficacy in chronic inflammatory arthritis in mice. METHODS: Tumor necrosis factor-transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF-C was cloned into an AAV expression vector to generate AAV-VEGF-C. The joints of TNF-Tg mice were injected with AAV-VEGF-C or AAV-luciferase (AAV-Luc) as a control. During the 4 months following injection, magnetic resonance imaging of the joints and lymphatic imaging were performed to assess changes in synovial volume and lymph flow from the joint tissues to local draining lymph nodes. Joint inflammation, bone erosion, and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry. RESULTS: Intraarticular administration of AAV-VEGF-C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in the joint sections, as compared with that in control AAV-Luc-injected joints, during the 4-month period. This was accompanied by a reduction in the area of inflammation, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from the joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates, and was significantly improved with AAV-VEGF-C treatment. CONCLUSION: Intraarticular injection of AAV-VEGF-C increased lymphangiogenesis and improved lymphatic drainage from the inflamed joints of mice, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.

Valproic acid decreases urothelial cancer cell proliferation and induces thrombospondin-1 expression.

BACKGROUND: Prevention of bladder cancer recurrence is a central challenge in the management of this highly prevalent disease. The histone deacetylase inhibitor valproic acid (sodium valproate) has anti-angiogenic properties and has been shown to decrease bladder cancer growth in model systems. We have previously shown reduced expression of thrombospondin-1 in a mouse model and in human bladder cancer relative to normal urothelium. We speculated that inhibition of angiogenesis by valproate might be mediated by this anti-angiogenic protein. METHODS: Bladder cancer cell lines UMUC3 and T24 were treated with valproate or another histone deacetylase inhibitor, vorinostat, in culture for a period of three days. Proliferation was assessed by alamar blue reduction. Gene expression was evaluated by reverse transcription of RNA and quantitative PCR. RESULTS: Proliferation assays showed treatment with valproate or vorinostat decreased proliferation in both cell lines. Histone deacetylase inhibition also increased relative expression of thrombospondin-1 up to 8 fold at 5 mM valproate. CONCLUSIONS: Histone deacetylase inhibitors warrant further study for the prevention or treatment of bladder cancer.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy.

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

A high-throughput semi-automated bone segmentation workflow for murine hindpaw micro-CT datasets.

INTRODUCTION: Micro-computed tomography (µCT) is a valuable imaging modality for longitudinal quantification of bone volumes to identify disease or treatment effects for a broad range of conditions that affect bone health. Complex structures, such as the hindpaw with up to 31 distinct bones in mice, have considerable analytic potential, but quantification is often limited to a single bone volume metric due to the intensive effort of manual segmentation. Herein, we introduce a high-throughput, user-friendly, and semi-automated method for segmentation of murine hindpaw µCT datasets. METHODS: In vivo µCT was performed on male (n = 4; 2-8-months) and female (n = 4; 2-5-months) C57BL/6 mice longitudinally each month. Additional 9.5-month-old male C57BL/6 hindpaws (n = 6 hindpaws) were imaged by ex vivo µCT to investigate the effects of resolution and integration time on analysis outcomes. The DICOMs were exported to Amira software for the watershed-based segmentation, and watershed markers were generated automatically at approximately 80% accuracy before user correction. The semi-automated segmentation method utilizes the original data, binary mask, and bone-specific markers that expand to the full volume of the bone using watershed algorithms. RESULTS: Compared to the conventional manual segmentation using Scanco software, the semi-automated approach produced similar raw bone volumes. The semi-automated segmentation also demonstrated a significant reduction in segmentation time for both experienced and novice users compared to standard manual segmentation. ICCs between experienced and novice users were >0.9 (excellent reliability) for all but 4 bones. DISCUSSION: The described semi-automated segmentation approach provides remarkable reliability and throughput advantages. Adoption of the semi-automated segmentation approach will provide standardization and reliability of bone volume measures across experienced and novice users and between institutions. The application of this model provides a considerable strategic advantage to accelerate various research opportunities in pre-clinical bone and joint analysis towards clinical translation.